Spatially barcoded microarray

ABSTRACT

The present disclosure provides a microarray comprising a plurality of probes. Each probe comprises a first oligonucleotide and a second oligonucleotide. The location of each probe on the microarray can be determined by the length of the first oligonucleotide and the length of the second oligonucleotide, thus providing a spatially barcoded microarray. Also provided are the methods of producing such spatially barcoded microarray. Also provided are the method of using such spatially barcoded microarray.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority to U.S. provisional patentapplication No. 63/213,681 filed Jun. 22, 2021, the entire disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to molecular biology and assays.More particularly, the invention relates to compositions and assays fordetermining spatial distributions of a large number of biologicalmolecules in a solid sample.

BACKGROUND OF THE INVENTION

The relationship between gene activities and where the activities occurwithin a tissue is critical to understanding normal development anddisease pathology. Spatial transcriptomics is a groundbreaking molecularprofiling technology that reveals both the RNA sequence and theirspatial locations in a tissue sample by capturing tissue RNA using aspatially barcoded microarray. Fabrication of a microarray withspatially barcoded capture probes is critical for the success of spatialtranscriptomics.

Currently, spatial barcoding is achieved by several technologies,including microspotting of nucleotides, array of split-pool-barcodedbeads, microfluidic channels, or in-situ solid-phase amplification.These spatial barcoding technologies, however, have several limitations.

The methods based on microspotting or microfluidic channels have limitedresolutions. The split-pool-barcoded beads method, though has a higherresolution, requires a decoding process which is time consuming and usesspecific costly equipment. The method based on in-situ solid-phaseamplification is complicated and expensive.

Therefore, there is a continuing need to develop new spatially barcodedmicroarrays that are less expensive, easy to fabricate, flexible in thearray dimension and resolution, and highly scalable.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a spatially barcodedmicroarray. In some embodiments, the spatially barcoded microarraycomprises:

-   -   a solid substrate having a surface; and    -   an array of N probes immobilized on the surface, wherein each        probe comprises a barcode region comprising a first barcode        oligonucleotide linked to a second barcode oligonucleotide,    -   wherein for any pair of the probes consisting of an ith probe        and a jth probe (1<<i<j<<N),        -   the ith probe comprises the first barcode oligonucleotide A,            linked to the second barcode oligonucleotide B_(i), and        -   the jth oligonucleotide comprises the first barcode            oligonucleotide A_(A) linked to the second barcode            oligonucleotide    -   wherein        -   the ith probe has a location of (X_(i), Y_(i)) under an X-Y            axis on the surface, and the jth oligonucleotide has a            location of (X_(j), Y_(j)) under the X-Y axis on the            surface,    -   wherein        -   if X_(i) is larger than X_(j), then the barcode            oligonucleotide A_(i) is longer than the barcode            oligonucleotide A_(j), and        -   if Y_(i) is larger than Y_(j), then the barcode            oligonucleotide B_(i) is longer than the barcode            oligonucleotide B_(j), and    -   wherein the length of the barcode oligonucleotides A_(i) and        B_(i), and the length of the barcode oligonucleotides A_(j) and        B_(j) identify the locations of the ith and the jth probes on        the surface, respectively.

In some embodiments, each probe further comprises a captureoligonucleotide.

In some embodiments, in each probe the first barcode oligonucleotide islinked to the 3′ end of the second barcode oligonucleotide. In someembodiments, in each probe the first barcode oligonucleotide is linkedto the 5′ end of the second barcode oligonucleotide.

In some embodiments, the sequence of the barcode oligonucleotide A_(i)comprises the sequence of the barcode oligonucleotide A_(j), or viceversa. In some embodiments, the sequence of the barcode oligonucleotideB_(i) comprises the sequence of the barcode tag B_(j), or vice versa.

In some embodiments, N is larger 100, 1,000, 10,000, 10,000, or1,000,000.

In some embodiments, each probe has a free 3′ end of a nucleotide.

In some embodiments, each probe further comprises a cleavage domain, afunctional domain, and a unique probe identifier, or a combinationthereof.

In another aspect, the present disclosure provides a method forgenerating a spatially barcoded microarray. In some embodiments, themethod comprises:

-   -   (a) providing (i) a solid substrate comprising a surface,        and (ii) a plurality of first oligonucleotides immobilized on        the surface;    -   (b) exposing the plurality of the first oligonucleotides to a        fluidic flow, wherein the fluidic flow comprises a first liquid        and a second liquid that flow along a first direction, wherein        the first liquid and the second liquid are immiscible with each        other and form an interface parallel to a first direction,        wherein the first liquid comprises a first enzyme capable of        removing one or more nucleotides from the first        oligonucleotides; and    -   (c) adjusting the relative proportion of the first liquid and        the second liquid in the fluidic flow to allow the number of the        first oligonucleotides that are exposed to the first liquid        changes along with time, wherein the number of the one or more        nucleotides that are removed from each of the first        oligonucleotides correlates with the timespan in which each of        the first oligonucleotides is exposed to the first liquid,        thereby generating a plurality of probes each having a segment        of the first oligonucleotides, wherein the length of the        segments of the first oligonucleotides forms a gradient        perpendicular to the first direction.

In some embodiments, the method further comprises adding a secondoligonucleotide to the free end of each probe. In some embodiments, thesecond oligonucleotide is added by a first ligase enzyme.

In some embodiments, the method further comprises exposing the pluralityof probes to a second fluidic flow comprises a third liquid and a fourthliquid that flow along a second direction, wherein the third liquid andthe fourth liquid are immiscible with each other and form an interfaceparallel to the second direction, wherein the third liquid comprises asecond enzyme capable of shortening the probes by removing one or morenucleotides from the second oligonucleotides. In some embodiments, thesecond direction and the first direction form an angle of 90° or 270°.

In some embodiments, the method comprises adjusting the relativeproportion of the third liquid and the fourth liquid in the fluidic flowto allow the number of the probes that are exposed to the third liquidchanges along with time, wherein the number of the one or morenucleotides that are removed from the second oligonucleotides correlateswith the timespan in which each probe is exposed to the third liquid,thereby generating a microarray spatially barcoded by the length of thesegment of the first oligonucleotide and the segment of the secondoligonucleotide comprised in each probe.

In some embodiments, none of the first oligonucleotides or the secondoligonucleotides is completed removed from any of the probes

In some embodiments, the first or second enzyme capable of shorteningprobe is an exonuclease. In some embodiments, the exonuclease isselected from Exonuclease I, Exonuclease III, Exonuclease V, ExonucleaseVII, Exonuclease VIII, Exonuclease T, T5 Exonuclease, T7 Exonuclease,Lambda Exonuclease, and a combination thereof.

In some embodiments, the method further comprises adding a targetcapture oligonucleotide to the free end of the second oligonucleotide.In some embodiments, the target capture oligonucleotide is added by asecond ligase enzyme.

In some embodiments, the microarray has one or more microfluidicchannels and wherein the flow rate of the first liquid and the flow rateof the second liquid are controlled by one or more pump modules.

In some embodiments, the first or third liquid is aqueous phase and thesecond or fourth liquid is organic phase.

In some embodiments, the method for generating a spatially barcodedmicroarray comprises:

a) providing (i) a solid substrate comprising a surface, and (ii) aplurality of first oligonucleotides immobilized on the surface;

(b) exposing the plurality of the first oligonucleotides to a firstconcentration gradient of a first enzyme, wherein the firstconcentration gradient of the first enzyme varies along a firstdirection; and

(c) removing one or more nucleotides from the first oligonucleotides bythe first enzyme, wherein the number of the one or more nucleotides thatare removed from the first oligonucleotides correlates with theconcentration of the first enzyme at the location of each probe on thesurface, thereby generating a plurality of probes each having a segmentof the first oligonucleotides, wherein the length of the segments of thefirst oligonucleotides forms a gradient perpendicular to the firstdirection.

In some embodiments, the method further comprises a secondoligonucleotide to the free end of each probe. In some embodiments, thesecond oligonucleotide is added by a first ligase enzyme.

In some embodiments, the method further comprises exposing the pluralityof probes to a second concentration gradient of a second enzyme capableof removing one or more nucleotides from the second oligonucleotides,wherein the second concentration gradient of the second enzyme variesalong a second direction. In some embodiments, the second direction andthe first direction form an angle of 90° or 270°.

In some embodiments, the number of the one or more nucleotides that areremoved from the second oligonucleotides by the second enzyme correlateswith the concentration of the second enzyme at the location of eachprobe on the surface.

In some embodiments, none of the first oligonucleotides or the secondoligonucleotides is completed removed from any of the probes.

In some embodiments, the first or second enzyme capable of shortening anoligonucleotide is an exonuclease. In some embodiments, the exonucleaseis selected from Exonuclease I, Exonuclease III, Exonuclease V,Exonuclease VII, Exonuclease VIII, Exonuclease T, T5 Exonuclease, T7Exonuclease, Lambda Exonuclease and a combination thereof.

In some embodiments, the method further comprises adding a poly-dToligonucleotide to the free end of the second oligonucleotide.

In another aspect, the present disclosure provides a method formeasuring a nucleic acid target in a sample. In one embodiment, themethod comprises:

-   -   contacting the sample with a spatially barcoded microarray,        wherein the spatially barcoded microarray comprises:        -   a solid substrate having a surface; and        -   an array of N probes immobilized on the surface, wherein            each probe comprises a capture region capable of specific            binding to the biological target and a barcode region,        -   wherein for any pair of the probes consisting of an ith            probe and a jth probe (1<<i<j<<N),            -   the barcode region of the ith probe comprises a first                barcode oligonucleotide A_(i) linked to a second barcode                oligonucleotide B_(i), and            -   the barcode region of the jth probe comprises a first                barcode oligonucleotide A_(j) linked to a second barcode                oligonucleotide B_(j),        -   wherein            -   the ith probe has a location of (X_(i), Y_(i)) under an                X-Y axis on the surface, and            -   the jth oligonucleotide has a location of (X_(j), Y_(j))                under the X-Y axis on the surface,        -   wherein            -   if X_(i) is larger than X_(j), then the barcode                oligonucleotide A_(i) is longer than the barcode                oligonucleotide A_(j), and            -   if Y_(i) is larger than Y_(j), then the barcode                oligonucleotide B_(i) is longer than the barcode                oligonucleotide B_(j);    -   allowing the probes to interact with the nucleic acid target;    -   extending the probes specifically binding to the nucleic acid        target to generate a plurality of extended products; and    -   sequencing the plurality of extended products to determine the        length of the first barcode oligonucleotide and the length of        the second barcode oligonucleotide, thereby identifying the        location of the nucleic acid target in the sample.

In some embodiments, the nucleic acid target is mRNA.

In some embodiments, the capture region has a sequence complementary tothe sequence of the nucleic acid target. In some embodiments, thecapture region hybridizes with the nucleic acid target. In someembodiments, the capture region capable of specific binding to thenucleic acid target is poly-dT.

In some embodiments, the method further comprises a step of amplifyingthe extended products before the sequencing step.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1 shows a spatially barcoded microarray according to an embodimentof the invention.

FIG. 2 shows a subset of the array area in a spatially barcodedmicroarray according to an embodiment of the invention.

FIG. 3 shows the structure of a probe on a spatially barcoded microarrayaccording to an embodiment of the invention.

FIG. 4 shows the setup to fabricate a spatially barcoded microarray,which includes a microfluidic channel with six inlets or outlets and aprobe array inside the channel, wherein all the oligonucleotides in theprobe array have an equal length.

FIG. 5 shows the method of fabricating a one-dimension spatiallybarcoded oligonucleotide array using the setup illustrated in FIG. 4.The first liquid and the second liquid form a clear and movableinterface in the microfluidic channel.

FIG. 6 shows that a one-dimension spatially barcoded oligonucleotidearray fabricated by the present method.

FIG. 7 shows the array after adding a second oligonucleotide to theprobes of the one-dimension spatially barcoded oligonucleotide array vialigation.

FIG. 8 shows the method to fabricate a spatially barcodedoligonucleotide array in the second dimension by changing the flowdirection.

FIG. 9 shows the flow chart of the process to fabricate a two-dimensionspatially barcoded microarray according to an embodiment of theinvention.

FIG. 10 shows a setup to fabricate four two-dimension spatially barcodedarrays simultaneously.

FIG. 11 shows a concentration gradient generator to fabricate aspatially barcoded array.

FIG. 12 shows the method of measuring mRNA in a tissue sample using aspatially barcoded microarray according to another embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

In the Summary of the Invention above and in the Detailed Description ofthe Invention, and the claims below, and in the accompanying drawings,reference is made to particular features (including method steps) of theinvention. It is to be understood that the disclosure of the inventionin this specification includes all possible combinations of suchparticular features. For example, where a particular feature isdisclosed in the context of a particular aspect or embodiment of theinvention, or particular claim, that feature can also be used, to theextent possible, in combination with and/or in the context of otherparticular aspects and embodiments of the invention, and in theinvention generally.

Where reference is made herein to a method comprising two or moredefined steps, the defined steps can be carried out in any order orsimultaneously (except where the context excludes that possibility), andthe method can include one or more other steps which are carried outbefore any of the defined steps, between two of the defined steps, orafter all the defined steps (except where the context excludes thatpossibility).

Where a range of value is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictate otherwise, between the upper and lower limitof that range and any other stated or intervening value in that statedrange, is encompassed within the disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the disclosure.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

It will be appreciated that for simplicity and clarity of illustration,where appropriate, reference numerals have been repeated among thedifferent figures to indicate corresponding or analogous elements. Inaddition, numerous specific details are set forth in order to provide athorough understanding of the embodiments described herein. However, theembodiments described herein can be practiced without there specificdetails. In other instances, methods, procedures and components have notbeen described in detail so as not to obscure the related relevantfunction being described. Also, the description is not to be consideredas limiting the scope of the implementations described herein. It willbe understood that descriptions and characterizations of the embodimentsset forth in this disclosure are not to be considered as mutuallyexclusive, unless otherwise noted.

Definition

The following definitions are used in the disclosure:

It is understood that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto a “bridge probe” is a reference to one or more bridge probes, andincludes equivalents thereof known to those skilled in the art and soforth.

As used herein, “associate” or “associating” means physically direct orindirect attachment. For example, the label probe can hybridize to oneor more bridge probe, which hybridizes to the target probe, whichhybridizes the target nucleic acid, thereby the label probe isassociated with the target nucleic acid.

The term “at least” followed by a number is used herein to denote thestart of a range beginning with that number (which may be a range havingan upper limit or no upper limit, depending on the variable beingdefined). For example, “at least 1” means 1 or more than 1. The term “atmost” followed by a number is used herein to denote the end of a rangeending with that number (which may be a range having 1 or 0 as its lowerlimit, or a range having no lower limit, depending upon the variablebeing defined). For example, “at most 4” means 4 or less than 4, and “atmost 40%” means 40% or less than 40%. When, in this specification, arange is given as “(a first number) to (a second number)” or “(a firstnumber)-(a second number),” this means a range whose lower limit is thefirst number and whose upper limit is the second number. For example, 4to 20 nucleotides means a range whose lower limit is 4 nucleotides, andwhose upper limit is 20 nucleotides.

The term “complementarity” refers to the ability of a nucleic acid toform hydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%>, 70%>, 80%>, 90%, and 100% complementary). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. “Substantially complementary” as usedherein refers to a degree of complementarity that is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids thathybridize under stringent conditions.

The term “comprises” and grammatical equivalents thereof are used hereinto mean that other components, ingredients, steps, etc. are optionallypresent. For example, an article “comprising” (or “which comprises”)components A, B, and C can consist of (i.e., contain only) components A,B, and C, or can contain not only components A, B, and C but also one ormore other components.

The term “hybridizing” refers to the binding, duplexing, or hybridizingof a nucleic acid molecule preferentially to a particular nucleotidesequence under stringent conditions. The term “stringent conditions”refers to conditions under which a probe will hybridize preferentiallyto its target subsequence, and to a lesser extent to, or not at all to,other sequences in a mixed population (e.g., a cell lysate or DNApreparation from a tissue biopsy). A “stringent hybridization” and“stringent hybridization wash conditions” in the context of nucleic acidhybridization (e.g., as in array, microarray, Southern or northernhybridizations) are sequence dependent, and are different underdifferent environmental parameters. An extensive guide to thehybridization of nucleic acids is found in, e.g., Tijssen LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I, Ch. 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays,” (1993)Elsevier, N.Y. Generally, highly stringent hybridization and washconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe. Verystringent conditions are selected to be equal to the Tm for a particularprobe. An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on an array or on a filter in a Southern ornorthern blot is 42° C. using standard hybridization solutions (see,e.g., Sambrook and Russell Molecular Cloning: A Laboratory Manual (3rded.) Vol. 1-3 (2001) Cold Spring Harbor Laboratory, Cold Spring HarborPress, NY). An example of highly stringent wash conditions is 0.15 MNaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes. Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example of a low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4×SSC to 6×SSC at 40° C. for 15 minutes.

The term “location” as used herein may refer to a two-dimensional regionor a three-dimensional region. Suitably a region can be of any size.Suitably the maximum size of the region may be determined by theproperties of the microarray and/or the particular tissue or substrateused in the method. Suitably a location of interest may be any region,suitably any region on a substrate. Suitably, a location of interest isa two-dimensional region. Suitably a location of interest may be between1 pm²-150 mm² in size, suitably between 1 pm²-1 mm² in size, suitablybetween 1 pm²-1,000,000 pm² in size, suitably between 1 pm²-200,000 pm²in size, suitably between 1 pm²-20,000 pm² in size, suitably between 1pm⁷-1000 pm² in size.

The term “nucleic acid” (interchangeable with the term “polynucleotide”)as used herein refers to any polymer formed of a plurality of nucleotidebases, wherein the bases may be comprised of canonical or non-canonicalbases, and wherein the backbone may be modified or unmodified, andwherein the nucleotides may be linked by conventional phosphodiesterbonds, or non-conventional bonds such as phosphorothioate bonds orchemical bonds. The polynucleotide can additionally comprisenon-nucleotide elements such as labels, quenchers, blocking groups, orthe like. The polynucleotide can be, e.g., single-stranded ordouble-stranded.

A “nucleic acid target” or “target nucleic acid” means a nucleic acid,or optionally a region thereof, that is to be detected. The targetnucleic acid can have a nucleic acid sequence existing in the nature orany sequence designed and generated by human. For example, the nucleicacid sequence can be a genomic sequence of a prokaryotic or eukaryoticspecies. A prokaryotic species includes, for example, bacteria. Aeukaryotic species includes, for example, a fungus, a plant, an animal,e.g., a mammal. In particular, the sequence of a target nucleic acid ofinterest can be found in public available databases, for example, thedatabase of National Center for Biotechnology Information. The targetnucleic acid can be single-stranded or double stranded. In certainembodiments, the target nucleic acid is a single stranded nucleotidepolymer. In certain embodiments, the target nucleic acid is asingle-stranded DNA or RNA (e.g., mRNA, siRNA, LncRNA). In certainembodiments, the target nucleic acid has 15 or more nucleotides, e.g.,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more nucleotides.

As used herein, a “nucleotide sequence” or “polynucleotide sequence” isa polymer of nucleotides (an oligonucleotide, a DNA, a nucleic acid,etc.) or a character string representing a nucleotide polymer, dependingon context. From any specified nucleotide sequence, either the givennucleic acid or the complementary nucleic acid sequence can bedetermined.

The term “oligonucleotide” is used herein to mean a linear polymer ofnucleotide monomers. As used herein, the term may refer tosingle-stranded or double-stranded forms. Monomers making up nucleicacids and oligonucleotides are capable of specifically binding to anatural polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like, toform duplex or triplex forms. Such monomers and their internucleosidiclinkages may be naturally occurring or may be analogs thereof, e.g.,naturally occurring or non-naturally occurring analogs.

As used herein, a “probe” is an entity that can be used in the detectionof a target molecule. Typically, a probe in the present disclosurerefers to a nucleic acid molecule, with or without modification. Theprobe can be both single-stranded and double-stranded nucleotidepolymers. Unless indicated otherwise, the probes described in thepresent application is a single-stranded nucleotide polymer.

The term “sample” as used herein refers to any sample having or suspectof having the target nucleic acid, including sample of biological tissueor fluid origin, obtained, reached, or collected in vivo or in situ.Exemplary biological samples include but are not limited to cell lysate,a cell culture, a cell line, a tissue, an organ, a biological fluid, andthe like. In certain embodiments, the sample is a solid sample. In someembodiments, the sample is a tissue.

The term “sequentially” means that the components, domains or regions ina polynucleotide or probe are juxtaposed in a 5′ end to 3′ end, or 3′end to 5′ end order. It is understood that the polynucleotide or probemay include additional nucleotide sequence in adjacent to eachcomponent, domain or region or between two components, domains orregions that does not interfere with the function of the polynucleotideor probe.

The term “substrate” refers to a mechanical support upon which materialmay be disposed to provide functionality, whether mechanical,biological, optical, chemical or other functionality. A substrate may beunpatterned or patterned, partitioned or unpartitioned. Molecules on asubstrate may be disposed in features or may be uniformly disposed onthe substrate surface.

Spatially Barcoded Microarray

Fabrication of a microarray with spatially barcoded capture probes iscritical for spatial transcriptomics. The current barcoding mechanism isbased on different combinations of nucleotide sequences in the barcodedregion of oligonucleotides, which has low resolution, or is complicatedand expensive. The present disclosure in one aspect provides amicroarray which is spatially barcoded based on the length of theoligonucleotides (i.e., the number of nucleotides). Such microarray hasthe advantages of low cost, easy to fabricate, high flexibility in thearray dimension and resolution, and high scalability.

An exemplary embodiment of the spatially barcoded microarray describedherein is illustrated in FIG. 1. Referring to FIG. 1, the spatiallybarcoded microarray 100 is composed of a substrate 101, which has asubstantially flat surface. In certain embodiments, the substrate is aglass slide.

Referring as to FIG. 1, immobilized on the surface of the substrate 101is an array of probes, which forms an array area 102. The details of asubset of the array area 102 are illustrated in the inset of FIG. 1. Asshown in the inset, a plurality of probes 103 are immobilized on thesurface of the substrate. Each probe comprises at least a segment ofoligonucleotide. In some embodiments, the probes are covalently linkedto the substrate.

Each probe 103 immobilized on the substrate 101 is spatially barcoded,i.e., the location of each probe is traceable or determinable. In someembodiments, each probe 103 comprises at least a segment ofoligonucleotide and is barcoded by the length of the oligonucleotide. Asprovided in detail below, the location of each probe 103 can bedetermined by the length of oligonucleotides comprised in the probe.

The array area 102 can form any pattern on the substrate 101. In someembodiments, as illustrated in FIG. 1, the plurality of probes arearranged such that the array area 102 forms a shape of square. Othershapes or patterns of arrangement, such as rectangular, circular, oval,triangular, are also contemplated. Notably, the location of each probeimmobilized on the substrate 101 can be expressed in a Cartesian (i.e.,x-y axis) coordination system. It can be understood that each probeimmobilized on the substrate 101 has a different location. In practice,however, depending on the resolution of the spatially barcodedmicroarray, a group of probes close to each other has the same barcode(e.g., comprising oligonucleotides of the same length) and can beunderstood as having the same location on the substrate 101.

FIG. 2 illustrates the barcoding mechanism of an exemplary example ofthe microarray disclosed herein. Referring to FIG. 2, a subset of thearray area comprises a plurality of probes (e.g., 210, 220, 230, 240 and250) immobilized on the surface of the substrate. Each probe has abarcode region comprising at least two segments of oligonucleotide. Forexample, referring to FIG. 2, probe 210 comprises oligonucleotides 211and 212; probe 220 comprises oligonucleotides 221 and 222; probe 230comprises oligonucleotides 231 and 232; probe 240 comprisesoligonucleotides 241 and 242; probe 250 comprises oligonucleotides 251and 252.

Each probe has a location on the substrate according to a Cartesiancoordination system on the surface of the substrate. Referring to FIG.2, probe 210 has a location (X₁, Y₁) under the Cartesian coordinationsystem; probe 220 has a location (X₂, Y₂); probe 230 has a location (X₃,Y₃); probe 240 has a location (X₄, Y₄). It can be understood that forthe purposes of spatially barcoding, probes having distinct locations onthe substrate should have distinct barcode, i.e., the identity of theprobes is determinable according to the locations of the probes. It canalso be understood, however, that a group of probes close to each other(form a cluster or group) may have the same barcode and can beconsidered as having the same location on the substrate. For example,probe 210 and probe 250 are two probes close to each other and have thesame barcode. For the purposes of barcoding, probe 210 and probe 250 areconsidered as having the same location on the substrate.

As illustrated in FIG. 2, each probe immobilized on the substrate canspatially barcoded based on the length of the two oligonucleotidescomprised in the probe. As a principle, the length of the firstoligonucleotide comprised in each probe varies according to the locationof the probe on the x-axis; and the length of the second oligonucleotidecomprised in the probe varies according to the location of the probe onthe y-axis. Thus, the combination of the information regarding thelength of the first and second oligonucleotides identifies the locationof the probe.

In one embodiment, the length of one oligonucleotide comprised in aprobe is longer if the probe locates further on the x-axis. As anexample, referring to FIG. 2, probe 220 and probe 240 locate furtherthan probe 210 and probe 230 on the x-axis (i.e., X₂>X₁; X₄>X₁; X₂>X₃;and X₄>X₃). Correspondingly, the length of oligonucleotide 221 andoligonucleotide 241 are longer than the length of oligonucleotide 211and oligonucleotide 231. On the hand, probe 210 and probe 230 havesubstantially the same position on the x-axis (i.e., X₁=X₃).Correspondingly, the length of oligonucleotide 211 is substantially thesame as the length of oligonucleotide 231. Similarly, the length ofoligonucleotide 221 is substantially the same as the length ofoligonucleotide 241.

In one embodiment, the length of the second oligonucleotide comprised inthe probe is longer if the probe locates further on the y-axis. As anexample, referring to FIG. 2, probe 230 and probe 240 locate furtherthan probe 210 and probe 220 on the y-axis (i.e., Y₃>Y₁; Y₄>Y₁; Y₃>Y₂;and Y₄>Y₃). Correspondingly, the length of oligonucleotide 232 andoligonucleotide 242 are longer than the length of oligonucleotide 212and oligonucleotide 2. On the other hand, probe 210 and probe 220 havesubstantially the same position on the y-axis (i.e., Y₁=Y₃).Correspondingly, the length of oligonucleotide 212 is substantially thesame as the length of oligonucleotide 222. Similarly, the length ofoligonucleotide 232 is substantially the same as the length ofoligonucleotide 242.

In can be understood that the combination of the length of the first andsecond oligonucleotides comprised in each probe give rise to a uniquebarcode of the probe that can identify the location of the probe. Forexample, referring to FIG. 2, by comparing the length of oligonucleotide241 with oligonucleotide 211 and comparing the length of oligonucleotide242 with oligonucleotide 212, it can be determined that probe 240locates further than probe 210 on both x-axis and y-axis. Morecomprehensively, the relative position of probes 210, 220, 230 and 240can be determined based on the information of the length ofoligonucleotides 211, 212, 221, 222, 231, 232, 241 and 242.

In certain embodiments, the length of the oligonucleotide comprised inthe probe is 1-100 nucleotides, 1-90 nucleotides, 1-80 nucleotides, 1-70nucleotides, 1-60 nucleotides, 1-50 nucleotides, 1-40 nucleotides, or1-30 nucleotides. In certain embodiments, the length of theoligonucleotide comprised in the probe is 2-100 nucleotides, 2-90nucleotides, 2-80 nucleotides, 2-70 nucleotides, 2-60 nucleotides, 2-50nucleotides, 2-40 nucleotides, or 2-30 nucleotides. In certainembodiments, the length of the oligonucleotide comprised in the probe is3-100 nucleotides, 3-90 nucleotides, 3-80 nucleotides, 3-70 nucleotides,3-60 nucleotides, 3-50 nucleotides, 3-40 nucleotides, or 3-30nucleotides. In certain embodiments, the length of the oligonucleotidecomprised in the probe is 4-100 nucleotides, 4-90 nucleotides, 4-80nucleotides, 4-70 nucleotides, 4-60 nucleotides, 4-50 nucleotides, 4-40nucleotides, or 4-30 nucleotides. In certain embodiments, the length ofthe oligonucleotide comprised in the probe is 5-100 nucleotides, 5-90nucleotides, 5-80 nucleotides, 5-70 nucleotides, 5-60 nucleotides, 5-50nucleotides, 5-40 nucleotides, or 5-30 nucleotides.

In certain embodiments, each probe has a free 3′ end of a nucleotide.

In certain embodiments, each probe further comprises cleavage domain, afunctional domain, a unique probe identifier, a mRNA capture domain, ora combination thereof. FIG. 3 illustrates the structure of a singleexemplary probe. Referring to FIG. 3, a single probe is asingle-stranded polynucleotide comprising sequentially from 5′ end to 3′end: a cleavage domain, a functional domain (e.g., for amplification andsequencing), a spatial barcode, a probe identifier, and mRNA capturedomain. The probe is attached to the substrate at its 5′ end. Thecleavage domain contains a sequence capable of being recognized by anendonuclease which can release the probe (together with an amplificationproduct generated thereof as discussed infra) from the substrate. Incertain embodiments, the functional domain contains sequences that canbe used to generate an amplification product for sequencing analysis(see infra for detailed discussion). In certain embodiments, the spatialbarcode comprises sequentially from 5′ end to 3′ end a segment of thefirst oligonucleotide and a segment of the second oligonucleotide asdisclosed supra. In certain embodiments, the probe identifier containsprobe-specific sequences that can be used to identify the sample in amultiplex sequencing reaction. In some embodiments, the mRNA capturedomain contains a sequence capable of hybridizing to a target nucleicacid which can be used as capture probes for the target nucleic acidfrom histological slides or individual cells for identifying theirspatial information in the application of spatial transcriptomics orsingle-cell sequencing. In some embodiments, the mRNA capture domaincontains a poly-dT oligonucleotide.

Method of Manufacture

The spatially barcoded microarray can be manufactured by the method knowin the art, such as microspotting.

In another aspect, the present disclosure provides a method ofmanufacturing the spatially barcoded microarray disclosed herein.Comparing to the method currently known in the art, the method disclosedherein has the advantages of low cost, easy to fabricate, highflexibility in the array dimension and resolution, and high scalability.

In general, the method of manufacture disclosed herein involves exposingan array of oligonucleotides immobilized on a substrate to an enzymecapable of removing one or more nucleotides from the oligonucleotides.The method involves controlling the number of nucleotides removed fromeach oligonucleotide based on the location of each oligonucleotide suchthat the length of the oligonucleotide after treatment of the enzymerepresents the location of the oligonucleotide on the substrate.

In one embodiment, the number of nucleotides removed from eacholigonucleotide can be controlled by adjusting the timespan in which theoligonucleotide is exposed to the enzyme. In one embodiment, the methodinvolves

-   -   (a) providing (i) a solid substrate comprising a surface,        and (ii) a plurality of first oligonucleotides immobilized on        the surface;    -   (b) exposing the plurality of the first oligonucleotides to a        first liquid, wherein the first liquid comprises a first enzyme        capable of removing one or more nucleotides from the first        oligonucleotides; and    -   (c) controlling the timespan each of the first oligonucleotides        is exposed to the first liquid, wherein the number of the one or        more nucleotides that are removed from each of the first        oligonucleotides correlates with the timespan in which each of        the first oligonucleotides is exposed to the first liquid,        thereby generating a plurality of probes each having a segment        of the first oligonucleotides.

In some embodiments, the timespan each of the first oligonucleotides isexposed to the first liquid increases in the direction of the solidsubstrate (e.g., along x-axis of a Cartesian coordination system,thereby the length of the segments of the first oligonucleotides forms agradient along the first direction.

In some embodiments, the method of generating a spatially barcodedmicroarray by controlling the timespan in which the oligonucleotide isexposed to the enzyme can be conducted in a microfluidic channel, whichcan be understood referring to an exemplary embodiment illustrated inFIGS. 4-6.

FIG. 4 shows an exemplary embodiment of the setup for manufacturing thespatially barcoded microarray. Referring to FIG. 4, the setup formanufacturing the spatially barcoded microarray includes a substratehaving a substantially flat surface on which an oligonucleotide array isimmobilized. In certain embodiments, all the oligonucleotidesimmobilized on the substrate have the same sequence or at least the samenumber of nucleotides. The method of immobilizing oligonucleotides on asubstrate is known in the art. For example, Yousefi H et al. teachesseveral coupling strategies to covalently attach single-stranded nucleicacids to various functional surfaces (Advanced Materials Interfaces(2018) 5: 1800659). See, also, Rashid J and Yusof N, The strategies ofDNA immobilization and hybridization detection mechanism in theconstruction of electrochemical DNA sensor: A review, Sensing andBio-Sensing Research (2017) 16: 19-31.

Referring to FIG. 4, the setup for manufacturing the spatially barcodedmicroarray has one or more microfluidic channels, through which liquidcan flow across the surface of the substrate. The microfluidic channelhas one or more inlets and outlets, through which liquid can flow in orout of the microfluidic channel. The microfluidic channel can beconnected to one or more pump modules, which can control the flow rateof the liquid in or out of the microfluidic channel.

As illustrated in FIG. 5, in one embodiment, the oligonucleotidesimmobilized on the substrate are exposed to a fluidic flow. The fluidicflow comprises a first liquid and a second liquid that is injected tothe microfluidic channel from two inlets to form a two-phase flow alonga first direction. The first liquid comprises a first enzyme (e.g., anexonuclease) capable of shortening the oligonucleotides by removing oneor more nucleotides from the oligonucleotides. The second liquid is anorganic solvent such as mineral oil, isophorone, 2-methyltetrahydrofuran(2-MTHF), or cyclopentyl methyl ether (CPME), which is immiscible withthe first liquid. The two liquids form a clear interface parallel to theflow direction without molecular diffusion.

The flow rate of the first liquid and/or the second liquid iscontinuously adjusted, to allow the interface of the two liquids movesin a second direction perpendicular to the first direction and movesfrom one boundary of the oligonucleotide array to another boundary ofthe oligonucleotide array as shown in FIG. 5. In this way, the number ofthe oligonucleotides that are exposed to the exonucleases (in the firstliquid) is gradually increased or decreased, resulting in differentreaction time for the oligonucleotides at different locations of thearray. Because the number of nucleotides that are cleaved from theoligonucleotides is correlated to the length of the reaction time, thisprocess produces an array of oligonucleotides with a length gradient inthe second direction (i.e., perpendicular to the first direction) asshow in FIG. 6, which results in a one-dimension spatially barcodedoligonucleotide array. By controlling the moving speed of the liquidinterface, no oligonucleotide is totally cleaved.

In one embodiment, the one-dimension spatially barcoded oligonucleotidearray is further processed to generate a two-dimension spatiallybarcoded microarray, which can be understood in reference to FIG. 7 andFIG. 8.

Referring to FIG. 7, in order to generate a two-dimension spatiallybarcoded microarray, a second oligonucleotide is added to the free endof each probe on the one-dimension spatially barcoded microarraydescribed above (each probe comprises a segment of the firstoligonucleotide after exonuclease treatment). In some embodiments, thesecond oligonucleotide can be added by using ligation enzyme. The secondoligonucleotide comprises different sequences or different types ofnucleotides from the first oligonucleotide, which makes the secondoligonucleotide distinguished from the first oligonucleotide by DNAsequencing technology.

FIG. 8 illustrates an exemplary method of generating a length gradientfor the second oligonucleotide along the first direction. Referring toFIG. 8, the first liquid and the second liquid are injected from twoinlets of the microfluidic channel to generate a two-phase flow in thesecond direction. By continuously adjusting the flow rate of the firstliquid and/or the second liquid, the interface of the two liquid movesalong the first direction and moves from one boundary of theoligonucleotide array to another boundary of the oligonucleotide arrayas shown in FIG. 8. As a result, a length gradient of the secondoligonucleotides is generated along the first direction (i.e.,perpendicular to the second direction). A two-dimension spatiallybarcoded microarray combining the first oligonucleotides and the secondoligonucleotides (see FIG. 2) is thus generated.

The major steps of manufacturing a two-dimension spatially barcodedmicroarray disclosed herein can be understood in the flow chart of FIG.9.

To scale up the array fabrication, a multiple-phase microfluidic flowcomprises one or more the first liquid and one or more the second liquidis created as shown in FIG. 10 to fabricate two or more two-dimensionspatially barcoded oligonucleotide arrays simultaneously. Referring toFIG. 10, in a setup of manufacturing a spatially barcoded microarray,first oligonucleotides are mobilized on a substrate to form fouroligonucleotide arrays. The oligonucleotides arrays are then exposed toa fluidic flow in a microfluidic channel comprising multiple inlets andoutlets. The fluidic flow comprises a first liquid that is injected tothe microfluidic channel from two inlets. The fluidic flow alsocomprises a second liquid that is injected to the microfluidic channelfrom a middle inlet between the two inlets from which the first liquidis injected. The first liquid comprises a first enzyme (e.g., anexonuclease) capable of shortening the oligonucleotides by removing oneor more nucleotides from the oligonucleotides. The second liquid is anorganic solvent which is immiscible with the first liquid. The twoliquids form a clear interface parallel to the flow direction withoutmolecular diffusion. As a result, a three-phase flow is formed along afirst direction.

The flow rate of the first liquid and/or the second liquid iscontinuously adjusted, to allow the interface of the liquids moves fromthe middle of the microfluidic channel to the side as shown in FIG. 10.In this way, the number of the oligonucleotides that are exposed to theexonucleases (in the first liquid) is gradually increased or decreased,resulting in different reaction time for the oligonucleotides atdifferent locations of the array. It can be understood that in the setupof FIG. 10, the oligonucleotides located closer to the edge of thesubstrate will have shorter length as they are exposed to the firstliquid for longer time. As a result, four one-dimension spatiallybarcoded oligonucleotide arrays are generated. Using the same strategy,the one-dimension spatially barcoded oligonucleotide arrays can befurther processes to generate four two-dimension spatially barcodedoligonucleotide arrays by injecting the first and second liquid to themicrofluidic channel from a different direction (e.g., from bottom totop as shown in FIG. 10).

In another aspect, as illustrated in FIG. 11, a concentration-dependentcleavage process can be used to create a two-dimension spatiallybarcoded oligonucleotide array. Referring to FIG. 11, concentrationgradients of exonucleases are generated via a microfluidic channel withconcentration generators. In this setup, the number of nucleotides thatare cleaved from the oligonucleotides is correlated to the concentrationof the exonucleases at the specific location. For example, when theconcentration of the exonuclease forms a gradient in a direction fromtop of FIG. 11 to the bottom, an array of oligonucleotides with a lengthgradient from the top to the bottom direction can be generated.

In certain embodiments, the probes in the microarray further comprise acleavage domain, a functional domain, a unique probe identifier, a mRNAcapture domain, or a combination thereof. It can be understood that suchprobes can be manufactured by sequentially adding relevant domains tothe probes during the manufacture process. For example, to manufacture aprobe as illustrates in FIG. 3 (which is a single-strandedpolynucleotide comprising sequentially from 5′ end to 3′ end: a cleavagedomain, a functional domain, a spatial barcode, a probe identifier, andmRNA capture domain), the manufacture process can start withimmobilizing a plurality of foundation oligonucleotides on thesubstrate, wherein each foundation oligonucleotide comprisessequentially from 5′ end to 3′ end the cleavage domain, the functionaldomain and the first oligonucleotide illustrated in FIG. 4. Thefoundation oligonucleotides immobilized on the substrate are thenprocessed to generate a one-dimension oligonucleotide array as describedabove. The second oligonucleotides are then added to the 3′ end of eachprobe of the one-dimension oligonucleotide array and processed togenerate a two-dimension oligonucleotide array as described above. Theprobe identifier and mRNA capture domain can then be added to the 3′ endof each probe of the two-dimension oligonucleotide array. The method ofadding relevant domains to the probe is known in the art, e.g., using anucleic acid ligase.

Method of Use

In another aspect, the present disclosure provides a method of using thespatially barcoded microarray described herein to measure a biologicaltarget (e.g., a nucleic acid target) in a sample. In one embodiment, themethod comprises: contacting the sample with a spatially barcodedmicroarray described herein, allowing the probes to interact with thebiological target; extending the probes specifically binding to thebiological target to generate a plurality of extended products; andsequencing the plurality of extended products to determine the length ofthe first barcode oligonucleotide and the length of the second barcodeoligonucleotide, thereby identifying the location of each extendedproduct in the sample.

The major steps of an exemplary method of measuring mRNA in a sample areillustrated in FIG. 12. Referring to FIG. 12, to measuring mRNA in atissue sample, the sample is placed in contact with a spatially barcodedmicroarray described herein. The tissue is then treated with chemicalsto permeabilize the cells and release the mRNA in the cells, such thatthe mRNA released from the cells interacts with the probes on themicroarray. The microarray is then threated with a reaction mix tosynthesize the cDNA using mRNA as a template and the probes as primers.The synthesized cDNA is then pooled and prepared for high-throughputsequencing analysis. In certain embodiments, the probes on themicroarray have a structure as illustrated in FIG. 3. In suchembodiments, the synthesized products can be dissociated from thesubstrate by cutting the cDNA synthesis products at the cleavage domainof the probes. The cleaved synthesis products are amplified andprocesses for high-throughput sequencing, e.g., in an Illuminasequencing by synthesis system.

The sequencing results provide information of the cDNA as well as thelength of the first oligonucleotide and the second oligonucleotide inthe barcode region of the probe. As the length of the firstoligonucleotide and the second oligonucleotide in the barcode region isassociated with the location of the probe on the microarray, thelocation of the RNA in the sample can be identified.

What is claimed is:
 1. A method for generating a spatially barcodedmicroarray comprising an array of barcoded probes, the methodcomprising: (a) providing (i) a solid substrate comprising a surface,and (ii) an array of N first initial oligonucleotides of the same length(N is an integer larger than 1,000) immobilized on the surface, whereinfor any pair of the first initial oligonucleotides consisting of an ithfirst initial oligonucleotide and a jth first initial oligonucleotide(1<<i<j<<N), the ith first initial oligonucleotide has a location of(X_(i), Y_(i)) under a Cartesian X-Y axis on the surface, and the jthfirst initial oligonucleotide has a location of (X_(j), Y_(j)) under theCartesian X-Y axis on the surface; (b) exposing the array of N firstinitial oligonucleotides immobilized on the surface to a fluidic flowthat flows along the Y axis on the surface, wherein the fluidic flowcomprises a first liquid and a second liquid that are immiscible witheach other and form an interface parallel to the Y axis on the surface,wherein the first liquid comprises an exonuclease, thereby removing oneor more nucleotides from each of the array of N first initialoligonucleotides; and (c) adjusting the relative proportion of the firstliquid and the second liquid in the fluidic flow to move the interfacealong the X axis on the surface, thereby generating an array of N firstbarcode oligonucleotides, each first barcode oligonucleotide generatedby removing one or more nucleotides from a first initialoligonucleotide, wherein for any pair of the first barcodeoligonucleotides consisting of an ith first barcode oligonucleotide anda jth first barcode oligonucleotide (1<<i<j<<N), the ith first barcodeoligonucleotide is generated from the ith first initial oligonucleotideand has the location of (X_(i), Y_(i)) under the Cartesian X-Y axis onthe surface, and the jth first barcode oligonucleotide is generated fromthe jth first initial oligonucleotide and has the location of (X_(j),Y_(j)) under the Cartesian X-Y axis on the surface, wherein if X_(i) islarger than X_(j), then the ith first barcode oligonucleotide is longerthan the jth first barcode oligonucleotide.
 2. The method of claim 1,further comprising adding a second initial oligonucleotide to each ofthe first barcode oligonucleotide, thus generating an array of Nintermediate probes.
 3. The method of claim 2, wherein the secondoligonucleotide is added by a first ligase enzyme.
 4. The method ofclaim 2, further comprising exposing the array of intermediate probes toa second fluidic flow that flows along the X axis on the surface,wherein the second fluidic flow comprises a third liquid and a fourthliquid that are immiscible with each other and form a second interfaceparallel to the X axis on the surface, wherein the third liquidcomprises a second exonuclease, thereby removing one or more nucleotidesfrom each second initial oligonucleotide.
 5. The method of claim 4,comprising adjusting the relative proportion of the third liquid and thefourth liquid in the fluidic flow to move the second interface along theY axis on the surface, thereby generating an array of N second barcodeoligonucleotides, each second barcode oligonucleotide generated byremoving one or more nucleotides from a second initial oligonucleotide,wherein for any pair of the second barcode oligonucleotides consistingof an ith second barcode oligonucleotide and a jth second barcodeoligonucleotide (1<<i<j<<N), the ith second barcode oligonucleotide isgenerated from the ith second initial oligonucleotide and has thelocation of (X_(i), Y_(i)) under the Cartesian X-Y axis on the surface,and the jth second barcode oligonucleotide is generated from the jthsecond initial oligonucleotide and has the location of (X_(j), Y_(j))under the Cartesian X-Y axis on the surface, wherein if Y_(i) is largerthan Y_(j), then the ith second barcode oligonucleotide is longer thanthe jth second barcode oligonucleotide.
 6. The method of claim 1,wherein the exonucleases is selected from Exonuclease I, ExonucleaseIII, Exonuclease V, Exonuclease VII, Exonuclease VIII, Exonuclease T, T5Exonuclease, T7 Exonuclease, Lambda Exonuclease, and a combinationthereof.
 7. The method of claim 5, further comprising adding a poly-dToligonucleotide to the free end of each of the second barcodeoligonucleotides.
 8. The method of claim 7, wherein the poly-dToligonucleotide is added by a second ligase enzyme.
 9. The method ofclaim 1, wherein each barcoded probe further comprises a cleavagedomain, a functional domain, and a unique probe identifier, or acombination thereof.
 10. The method of claim 1, wherein the array of Nfirst initial oligonucleotides is exposed to the fluidic flow in amicrofluidic channel and wherein the flow rate of the first liquid andthe flow rate of the second liquid are controlled by one or more pumpmodules.
 11. The method of claim 5, wherein the first or third liquid isaqueous phase and the second or fourth liquid is organic phase.
 12. Themethod of claim 1, wherein each of the first initial oligonucleotides isimmobilized on the surface via the 5′-end.